Sl/Sld mice have an increased number of gap junctions in their bone marrow stromal cells

To study the defect of the hematopoietic inductive microenvironment (HIM) in Sl/Sld mice, femoral bone marrow tissue of 10 of each mutant, (Sl/Sld and W/Wv) their normal littermates (Sl+/Sl+ and W+/W+), and 20 normal C57BL mice were examined by electron microscopy using morphometric and statistical methods. Gap junctions were observed in all strains of mice, in the following stromal cell types: 1) reticular cells, 2) between reticular cells and periarterial adventitial cells, and 3) between periarterial adventitial cells. The frequency of gap junctions in bone marrow stromal cells of Sl/Sld mice (mean = 2.2/9.4 X 10(-3) mm2) was significantly higher than in control mice. It is suggested that there is a relationship between the increased numbers of gap junctions in bone marrow stromal cells of Sl/Sld mice and the defect in HIM function in these genetically anemic animals.     

Recombinant murine steel factor stimulates in vitro production of granulocyte-macrophage progenitor cells.

The ability of murine Steel factor to promote the in vitro production of granulocyte-macrophage progenitor cells (CFU-GM) was examined in short-term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL-3. CFU-GM responsive to GM-CSF, IL-3, and CSF-1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CFU-GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was potentiated by the addition of IL-3. Production of CFU-GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF-1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild-type protein in its capacity to promote CFU-GM production from normal bone marrow.     

Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.     

Functionally abnormal stromal cells and megakaryocyte size, ploidy, and ultrastructure in Sl/Sld mice

The first goal of the present studies was to determine if Sl/Sld megakaryocytes have features in common with the macrocytic megakaryocytes that genetically normal mice produce in response to acute platelet depletion. The second was to test the hypothesis that megakaryocyte abnormalities in Sl/Sld mice are due to genetically determined hemopoietic stromal cell abnormalities. Sizes and ploidies of mature Sl/Sld megakaryocytes were measured. Macrocytosis and a shift to higher ploidy values were found compared with normal. Within ploidy groups 16N-64N, Sl/Sld megakaryocytes were larger than normal megakaryocytes of the same ploidy. Transmission electron microscopy revealed that Sl/Sld megakaryocyte nuclei contain more and larger nucleoli, and the chromatin was more dispersed than in normal megakaryocyte nuclei of comparable maturity. Asynchronous megakaryocyte cytoplasmic maturation was found. Sl/Sld macrophages were also ultrastructurally abnormal. Megakaryocytic macrocytosis was reproduced in long-term bone marrow cultures in which the adherent layer was formed by Sl/Sld cells. It was the same if cultures were recharged with Sl/Sld or +/+ hemopoietic cells. Previously reported ambiguities in mixed cell cultures were avoided by recharging the adherent layers with only a million cells. These results were correlated with previously published observations. Sl/Sld megakaryocytes have features in common with megakaryocytes from acutely thrombocytopenic animals. One feature, macrocytosis, appears to be due to abnormal Sl/Sld stromal cells that are reproduced as adherent layer cells in long-term cultures. The responsible stromal cells in Sl/Sld mice may be counterparts of megakaryocytopoietic regulatory cells in the marrow stroma of normal animals.     

Haemopoietic stem cell proliferation in the bone marrow of S1/S1d mice

In marrow from Sl/Sld mice (but not +/+ mice) day 7 and day 8 CFU-S proliferate whilst day 10 and day 12 CFU-S exhibit negligible proliferation. Media conditioned by both +/+ and Sl/Sld marrow contains an inhibitor of CFU-S proliferation but day 8 CFU-S in +/+ and Sl/Sld marrow show marked dose-response differences to this factor. To inhibit the proliferation of Sl/Sld CFU-S required approximately ten times the concentration of inhibitor that inhibited the proliferation of +/+ CFU-S. Thus abnormally responsive day 8-CFU-S were shown to proliferate in an inhibitory environment. Abnormalities in Sl/Sld CFU-S function were also demonstrated in heterotopic transplantation experiments using +/+ and Sl/Sld donors and hosts to obtain ectopic bone marrow with various stromal (donor) and haemopoietic (host) combinations. Day 8 Sl/Sld CFU-S were seen to proliferate, irrespective of whether the stromal environment was derived from Sl/Sld or +/+ marrow. Sl/Sld mice are generally regarded as animals in which there is a genetically determined defect in haemopoiesis due to an abnormality in the haemopoietic environment. It is difficult, however, to attribute the abnormal CFU-S behaviour in these experiments to environmental factors and the results are consistent with mutation at the Sl locus affecting the responses of CFU-S to regulatory signals, i.e. the genetic defect is not confined to the stromal environment.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

Regulatory mechanisms of mast cell differentiation

Mast cells are a progeny of the multipotential hematopoietic stem cell. Most of progenies of the stem cell complete their differentiation within the bone marrow, but precursors of mast cells leave the bone marrow, migrate in blood, and invade into tissues. After the invasion, precursors proliferate and differentiate into mast cells. An appreciable proportion of mast cells retain proliferative potential after differentiation, and even after degranulation, some mast cells can proliferate and recover the original morphology. Proliferation of mast cells are regulated by both T cell-derived factors (i.e., IL-3 and IL-4) and fibroblast-derived factor(s). Mice of either W/Wv or Sl/Sld genotype lack mast cells, but mast cells do develop when bone marrow cells of W/Wv or Sl/Sld mice were cultured in the presence of T cell-derived factors. Mast cells derived from W/Wv mice cannot respond fibroblast-derived factor(s) and fibroblasts derived from Sl/Sld mice cannot support mast cells of normal mouse origin. Phenotypes of mast cells are determined by the environment in which the mast cells differentiated. However, when mast cells are transplanted into a new environment which is different from the original one, the mast cells acquire the phenotype which are dependent on the second environment.     

Bone marrow stromal proteoglycan heterogeneity: phenotypic variability between cell lines and the effects of glucocorticoid

Hematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component. Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation. Proteoglycan synthesis in the hematopoietic-supportive D2XRII, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S-sulfate precursor labelling and ion-exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC. The effects of glucocorticoid were also investigated. A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation. All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio-Sil TSK-400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media. The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers. CS/DS species were the major proteoglycans in all cultures. Hydrocortisone-free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell-associated form (HS1), partially excluded from the TSK-400 column, and a secretory form (HS2), eluting at Kd = 0.15. D2XRII cells also secreted an apparently-unique, high-density proteoglycan, Kd = 0.65, into the culture medium. Hydrocortisone at 10(-6) M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.     

Adhesion of mouse mast cells to fibroblasts: adverse effects of steel (Sl) mutation

Mouse bone marrow-derived cultured mast cells proliferate on +/+ mouse embryo-derived 3T3 fibroblasts, but not on Sl/Sld mouse embryo-derived 3T3 fibroblasts, in the absence of IL-3 and IL-4 (Fujita et al: Proc. Natl. Acad. Sci. U.S.A. 86:2888-2891, 1989). To further characterize the mast cell-fibroblast interactions and the effects of Sl mutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 10(3) to 5 x 10(5) cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited by an RGD-containing peptide, an anti-LFA-1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived from Sl/Sld mouse embryos. Adhesion to +/+ mouse spleen-derived fibroblasts lacking mast cell-supporting activity was comparable to that to Sl/Sld/3T3 cells. The failure of mast cells to adhere to fibroblasts with the Sl mutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild type Sl gene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analysis of these interactions.     

Lithium stimulation of HPP-CFC and stromal growth factor production in murine Dexter culture.

We have previously reported that the addition of lithium chloride (LiCl) to murine Dexter cultures results in increased numbers of progenitor and mature hematopoietic cells of the granulocyte, macrophage, and megakaryocyte lineages. We now report the effect of various levels of LiCl on the high proliferative potential colony-forming cell (HPP-CFC) in Dexter culture and on the induction of growth factors from Dexter stromal cells. LiCl (4 mEq/L) stimulated supernatant HPP-CFC for the first 4 weeks of culture (150-275%), and stimulated stromal HPP-CFC at week 3 (170-222%). Higher levels of lithium (8 and 12 mEq/L) selectively stimulated supernatant HPP-CFC, macrophage, and eosinophil production, whereas granulocytes and granulocyte-macrophage colony-forming cells (CFU-C) were inhibited. mRNA expression was evaluated from week 4 Dexter cultures that received a pulse or continuous exposure to lithium and had received either 0 or 1,100 cGy irradiation. Four mEq/L LiCl stimulated increased expression of G-CSF, GM-CSF, IL-6, and, in the nonirradiated stroma continuously exposed to lithium, CSF-1 mRNA. In general, the higher levels of lithium stimulated increased mRNA expression for these same growth factors. mRNA for the recently described Steel factor was decreased with increasing levels of lithium added to either normal or irradiated stroma. Bioassays of conditioned medium (cm) from irradiated cultures against the FDC-P1 and T1165 cell lines indicated cytokine activity, which was blocked by antibodies to GM-CSF and IL-6, respectively. Altogether these data show that lithium stimulates Dexter HPP-CFC, and this stimulation appears to be mediated by multiple growth factors that are induced from stromal cells.     

Stromal cells provide signals different from cytokines for STAT5 activation in hematopoietic cells

After detachment from the stromal cells, hematopoietic stem cells are thought to differentiate to the cytokine-dependent stages where their growth and differentiation are promoted by these cytokines. To examine the stromal regulation of hematopoietic stem cells, we previously established a primitive hematopoietic stem-like cell line, THS119, whose growth was dependent on the bone marrow stromal cell line, TBR59, and from which IL-3- (THS119/IL-3) or IL-7- (THS119/IL-7) dependent cell lines were then generated. Using these cell lines, we examined the difference in signals mediated by the stromal cells and cytokines. The cytokine-dependent cell lines (THS119/IL-3 and THS119/IL-7) showed induction of STAT5 phosphorylation and target genes for STAT5 such as CIS, pim-1, p21 and bcl-xL upon addition of IL-3 or IL-7. IL-3 or IL-7 also induced STAT5 phosphorylation and STAT5 target genes of the stromal cell-dependent cell line, THS119, in the absence of stromal cells at levels similar to the cytokine-dependent cell lines. However, quite interestingly, TBR59 stromal cells could not induce STAT5 phosphorylation of THS119 cells, although they did induce STAT5 target genes in THS119 cells. In addition, the mRNAs for STAT5 target genes in THS119 cells on the stromal cells seemed to be more stable than those in the cytokine-dependent cell lines. Expression of the antiapoptotic genes bcl-2 and bcl-xL was higher in the stromal cell-dependent cell line than in the cytokine-dependent cell lines. These results suggested that stromal cells and cytokines may provide different signals for growth and differentiation of the hematopoietic cells.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

An allelic series of mutations in the Kit ligand gene of mice. II. Effects of ethylnitrosourea-induced Kitl point mutations on survival and peripheral blood cells of Kitl(Steel) mice

The ligand for the Kit receptor tyrosine kinase is Kit ligand (Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is encoded at the Steel (Sl) locus of mice. Previous studies revealed that Kitl(Sl) mutations have semidominant effects; mild pigmentation defects and macrocytic, hypoplastic anemia occur in heterozygous mice, and more severe pigmentation defects and anemia occur in homozygotes. Lethality also occurs in mice homozygous for severe Kitl(Sl) mutations. We describe the effects of seven new N-ethyl-N-nitrosourea (ENU)-induced Kitl(Sl) mutations and two previously characterized severe Kitl(Sl) mutations on pigmentation, peripheral blood cells, and mouse survival. Mice heterozygous for each of the nine mutations had reduced coat pigmentation and macrocytosis of peripheral blood. In the case of some of these mutations, however, red blood cell (RBC) counts, hemoglobin concentrations, and hematocrits were normal in heterozygotes, even though homozygotes exhibited severely reduced RBC counts and lethality. In homozygous mice, the extent of anemia generally correlates with effects on viability for most Kitl(Sl) mutations; i.e., most mutations that cause lethality also cause a more severe anemia than that of mutations that allow viability. Interestingly, lethality and anemia were not directly correlated in the case of one Kitl(Sl) mutation.     

Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci.

Mutations in either the dominant white-spotting (W) or Steel (Sl) loci of the mouse lead to coat color, primordial germ cell and hematopoietic defects. Consistent with the cell autonomous and microenvironmental nature of W and Sl mutations, respectively, it has recently been shown that W encodes the c-kit receptor tyrosine kinase while Sl encodes a ligand for this receptor. Previous in situ hybridization analysis has shown that both c-kit and steel are expressed in the embryo in anatomical sites known to be affected by W and Sl mutations and in various tissues in which no corresponding phenotype has been described. To investigate the possible involvement of the Kit transduction pathway in developmental processes, we compared the patterns of expression of c-kit and steel in wild-type embryos and in embryos homozygous for severe (lethal) and mild (viable) alleles at the W and Sl loci. In addition, we analyzed the patterns of expression of both genes in adult wild-type and mutant gonads and brain. Both c-kit and steel are contiguously expressed in a wide variety of anatomical locations in both the developing embryo and in the adult. In adult gonads, steel is expressed in the follicular cells of the ovary and in Sertoli cells of the testis, the layers that immediately surround the c-kit expressing germ cells. In adult brain, the complementary patterns are particularly striking in the olfactory bulb, cerebral cortex, hippocampus region and cerebellum. steel expression in brain is probably restricted to neurons in certain areas, while c-kit is expressed in neurons and in some glial cells. Severe mutations in the W or Sl loci result in dramatic reduction or absence of c-kit positive cells in lineages known to be affected by these mutations. In contrast, these mutations do not affect the number or histological organization of c-kit positive cells in the embryonic peripheral or central nervous systems, nor is the number or organization of c-kit positive cells detectably altered in Wv/Wv or Sld/Sld adult brain. Taken together, these results suggest that the Kit signaling pathway is not obligatory for the viability and/or migration of most c-kit expressing cells either because of functional redundancy with another signaling pathway or because the Kit pathway is involved in post-developmental processes of mature cells.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1α induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1α induced IL-1α and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34⁺ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment. J. Cell. Physiol. 176:57–66, 1998. © 1998 Wiley-Liss, Inc.     

Oncostatin M regulates mesenchymal cell differentiation and enhances hematopoietic supportive activity of bone marrow stromal cell lines

Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.     

Effects of Endogenous Epidermal Growth Factor Receptor Signaling on DNA Synthesis and ERK Activation in a Cytokine-Dependent Hematopoietic Cell Line

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5.12. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic cells.     

Isolation and characterization of proteoglycans synthesized by rat myoblasts L6J1 in culture

Proteoglycans synthesized by rat myoblasts L6J1 in culture were isolated using sorbent Q-Sepharose from culture medium, extracellular matrix (ECM), and cells. Elution of the sorbed material in a NaCl gradient separated proteoglycans from the bulk of proteins eluted at low concentration of the salt. Four fractions (fractions I-IV) were obtained for each component of the cell culture, including two proteoglycan fractions for the ECM and culture medium and one fraction for the myoblasts. Proteoglycans of the culture medium were virtually completely represented by proteoglycans of fetal calf serum. With enzymes chondroitinase ABC and heparinase III chondroitin/dermatan sulfate proteoglycans were shown to prevail in all components of the myoblast culture. The core proteins of proteoglycans were characterized by electrophoresis.     

Gametic and pleiotropic defects in mouse fetuses with Hertwig''s macrocytic anemia

Pleiotropic effects on germ cell number, hematologic status, and body size are described in 12- to 15-day WBB6F1 normal (+/-) and defective (an/an) mouse fetuses, with special emphasis on gametogenesis. Differences between genotypes were apparent by Day 12. At 12 days, normal testes contained many germ cells and frequent normal mitoses, and the number of germ cells increased rapidly from Day 12 to Day 15. By contrast, 12-day an/an testes contained fewer germ cells, frequently degenerating, and many abnormal mitoses. Their number of germ cells decreased rapidly, so that almost none persisted to Day 15. Normal ovaries contained many germ cells, with much normal mitosis on Day 12 and 13, followed by meioses, but the smaller an/an ovaries contained few germ cells, with little mitosis, some meiosis, and very much degeneration. The erythrocyte counts of both normal and anemic fetuses increased approximately fourfold between 12 and 15 days, but at comparable ages, total counts were always lower in an/an fetuses than in normal littermates. At all ages, Hertwig's anemic (an/an) fetuses were somewhat smaller than their normal littermates. Although both W/Wv and Sl/Sld mice also show macrocytic anemia and germ cell failure, the great difference in etiology of their germ cell defects indicates that an/an gene action must be qualitatively different from that in either W/Wv or Sl/Sld mice.